Although adjuvant platinum-based chemotherapy (AC) has been shown to improve survival of patients with completely resected stage II and stage IIIA non-small cell lung cancer (NSCLC), its effect is limited. Nestin is a class VI intermediate filament protein expressed in neural stem cells and several cancer cells including NSCLC. In the present study, we aimed to determine its prognostic significance concerning survival in NSCLC patients receiving AC.Nestin expression in cancer cells was immunohistochemically studied in 90 patients with completely resected stage II and stage IIIA NSCLC treated with AC and its association with clinicopathologic parameters, including ABCG2, E-cadherin, and vimentin expression, was evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of nestin expression on survival.P = 0.006) and vimentin positive expression (P < 0.001). In survival analysis, nestin expression was significantly associated with a poorer prognosis (P = 0.028). Multivariable analysis confirmed that nestin expression is an independent prognostic indicator in NSCLC patients receiving AC .Nestin expression was observed in 28 of the 90 (31.1%) NSCLCs. Clinicopathologically, nestin expression was associated with loss of E-cadherin expression (The present study reveals that nestin expression is a prognostic indicator of a poorer survival probability in NSCLC patients receiving AC, although its prognostic significance still requires confirmation with larger patient populations. Primary lung cancer is the leading cause of cancer mortality worldwide . While sNestin is a class VI intermediate filament protein that is specifically expressed in stem/progenitor cells of the developing central nervous system . Nestin The study was approved by the Ethics Committee of the Kitasato University School of Medicine (B15-74) and followed the Declaration of Helsinki protocol. All patients were approached based on approved ethical guidelines, agreed to participate in this study, and could refuse entry and discontinue participation at any time. All participants proved written consent.A total of 90 consecutive NSCLC patients with completely resected stage II and stage IIIA treated with AC from January 2003 to September 2012 at the Kitasato University Hospital were included in this retrospective cohort study. Eleven of 90 patients were also included in our previous study . PatientTen-percent formalin-fixed and paraffin-embedded tissues were processed into 3-μm-thick sections. The sections were reacted with 100-times-diluted anti-nestin antibody  for 2 hrs at room temperature. The details of the procedure were described previously from our laboratory .For ABCG2, E-cadherin, and Vimentin immunohistochemical staining, for antigen retrieval, the sections were autoclaved in 0.01 mol/L citrate buffer (pH 6.0) with 0.1% Tween 20 at 121°C for 10 min. The sections were reacted with 200-times-diluted anti-ABCG2 antibody  and anti-Vimentin antibody  for 2 hrs at room temperature, and 200-times-diluted anti-E-cadherin antibody  for 1 hr at room temperature.For nestin and ABCG2, cytoplasmic immunostaining in tumor cells was considered to be positive. The stainability of peritumoral vascular endothelial cells in nestin and bronchial epithelial cells in ABCG2 was used as an internal positive control. The staining intensity was categorized into 4 groups by comparing the staining intensity of internal positive control: 0 = negative, 1 (weak) = weaker than positive control, 2 (moderate) = the same as positive control, and 3 (strong) = stronger than positive control. Tumors with a staining score of 2 or 3 were judged as positive. Immunohistochemical staining was semi-quantitatively assessed based on the approximate percentage of positive cells over the total number of tumor cells and was determined according to the criteria described previously as follows: nestin negative: <5%, nestin positive: ≥5% ; ABCG2 nFor E-cadherin and vimentin, cell membrane immunostaining in tumor cells was considered to be positive in E-cadherin, and cytoplasmic immunostaining in tumor cells was considered to be positive in vimentin. According to previous studies , 13, eva2 test or Fisher’s exact test, as appropriate. DFS and OS of patients was estimated by using the Kaplan-Meier method and compared by the log-rank test. A multivariable analysis was estimated by using the Cox proportional hazards model. If the P-value was less than 0.05, differences were considered significant. All reported P-values are two-sided.All statistical analyses were performed using SPSS version 23.0 software . In immunohistochemical analysis, inter-rater reliability between two investigators was calculated using the kappa statistic. The kappa coefficient was interpreted according to the following coefficient proposed by Landis and Koch: ≤ 0.20 (slight agreement); 0.21–0.40 (fair); 0.41–0.60 (moderate); 0.61–0.80 ; and 0.80–1.00  . The relThe clinicopathological characteristics of the patients are summarized in Cytoplasmic nestin expression in tumor cells was observed in 28 of 90 (31.1%) NSCLCs . These wInter-rater reliability between two investigators on nestin expression was in almost perfect agreement with the kappa coefficient of 0.872. Inter-rater reliabilities between two investigators on ABCG2, E-cadherin, and vimentin expressions were in substantial agreement with the kappa coefficients of 0.712, 0.755, and 0.779, respectively.P = 0.18). However, nestin expression was significantly associated with loss of E-cadherin expression (P = 0.006) and positive expression of vimentin (P < 0.001). Nestin expression and EMT phenotype according to expression of E-cadherin and vimentin are showed in P < 0.001). In the present study, 31 patients were analyzed for EGFR mutation status. EGFR common mutation (Del 19 or L858R) was detected in 20 of 31 (64.5%) patients. These were then divided into 4 of the 7 (57.1%) nestin-positive group and 16 of the 24 (66.7%) nestin-negative group, and the status of EGFR mutation was not different between nestin-positive and nestin-negative groups (P = 0.67).The relationships between nestin expression and clinicopathologic characteristics are summarized in P = 0.256, All the patients were included in the survival analysis. The overall follow-up periods ranged from 5.8 to 149.7 months , the median DFS for all patients was 41.0 months , 27.0–54.9). The median DFS was not significantly different, with 22.3 months for patients with nestin-positive tumors and 42.3 months for those with nestin-negative tumors .The median OS for all patients was 114.5 months . The median OS was 49.9 months for patients with nestin-positive tumors and 114.5 months for those with nestin-negative tumors, and nestin expression was significantly associated with a poorer prognosis  in AC-naïve cohort. It may further emphasize that nestin is a marker for chemoresistance. Although nestin expression was not a significant predictive factor for DFS, nestin-positive cases appear to have recurred earlier than nestin-negative cases in the relative early phase after surgery (for instance at 24 months). According to these results, nestin expression may influence chemoresistance in patients with recurrent NSCLC after surgery. In the treatment of patients with postoperative recurrence, patients with EGFR mutations have a significantly longer survival than those with wild type EGFR when treated with EGFR-TKIs [The aim of AC in patients with resected NSCLCs is to eradicate micrometastasis and improve their survival. In the present study, we focused on the patients with completely resected stage II and IIIA NSCLC treated with AC, and estimated the prognostic impact of nestin expression on survival. We have demonstrated that nestin expression appears to be associated with poorer prognosis and is an independent prognostic factor for survival in patients receiving AC. In addition, we evaluated the prognostic impact of nestin on survival in 59 cases (8 nestin-positive cases) with AC-naïve, completely resected stage II and IIIA NSCLCs in populations of our previous study  and nestGFR-TKIs . In the To investigate the relationship between nestin expression and chemoresistance, we further examined ABCG2- and EMT-related marker expression. ABCG2, also known as breast cancer resistant protein, is a transporter protein and plays a major role in chemoresistance . Lechnerin vitro and in vivo include the acquisition of a spindle-like/fibroblastic morphology, the gain of mesenchymal markers , and the loss of epithelial cell surface markers and cytoskeleton components  [Previous studies reported that EMT play a role in cancer progression and drug resistance . The haleratins) . Zhao eteratins) . Therefoeratins) . Mochizueratins) . In surgeratins) . In the eratins) . SeveralThere are a few limitations to our study. First, the sample size of our study is relatively small, and the follow-up duration is relatively short. Therefore, a larger population and longer follow-up duration will be needed in future to clarify the findings of the present study. Second, 32.3% of patients were treated with a carboplatin (CBDCA)-based regimen in the present study. The Lung Adjuvant Cisplatin Evaluation meta-analysis demonstrated that adjuvant cisplatin (CDDP)-based chemotherapy is of benefit in completely resected NSCLC . HoweverWe revealed that nestin expression is significantly associated with EMT-related markers such as loss of E-cadherin and gain of vimentin, and is a prognostic indicator of a poorer survival probability in NSCLC patients receiving AC, although its prognostic significance still requires confirmation with larger patient populations. Further understanding of the links between nestin, EMT, and chemoresistance may lead to the identification of novel therapeutic targets for the prevention of resistant to conventional chemotherapy in NSCLC.


Mean weight loss did not differ between groups (−2.7 ± 2.6 and −3.2 ± 3.0 kg for CON (n = 11) and HPR (n = 10) respectively, p = 0.801); however, the 6-week change in fat-free mass (FFM) differed significantly between groups . IR improved in HPR vs. CON participants . The change in HOMA-IR was related to the change in FFM among participants . Thus, a high-protein diet using novel functional foods combined with modest calorie restriction was 140% more effective for reducing HOMA-IR in healthy adults compared to a lower protein, standard diet with an equal level of calorie restriction.Significant reductions in insulin resistance (IR) can be achieved by either calorie restriction or by the increase of lean mass. However, calorie restriction usually results in significant loss of lean mass. A 6-week randomized controlled feeding trial was conducted to determine if a calorie-restricted, high-protein diet (~125 g protein/day consumed evenly throughout the day) using novel functional foods would be more successful for reducing IR in comparison to a conventional diet (~80 g protein/day) with a similar level of calorie restriction. Healthy adults (age 20–75 years; body mass index, 20–42 kg/m Insulin resistance (IR) is characterized by the decreased ability of insulin to exert its metabolic actions on target cells. The molecular mechanisms of this failure are complex, but appear to be intimately linked with increased inflammation ,2,3. SigIR is also modified by a change in lean mass relative to total mass in the absence of weight loss, and retention of lean mass over time predicts reductions in IR . It is wHence, a calorie-restricted high-protein diet, with protein distributed evenly throughout the day may represent a particularly effective strategy for reducing IR under conditions of calorie restriction. This single-blinded, randomized controlled weight loss trial compared the efficacy of a standard, calorie-restricted control diet versus a protein-rich, calorie-restricted diet containing novel functional food products consumed at each meal daily, for reducing homeostasis model assessment-estimated insulin resistance (HOMA-IR) while simultaneously increasing fat-free mass (FFM) in adults at risk for IR.High-quality protein pasta (orzo and fusilli) enriched with whey protein isolate, wheat gluten and egg white protein (% energy: 43/27/30 for carbohydrate/protein/fat) and high-protein flaked breakfast cereal enriched with wheat gluten (% energy: 35/30/35 for carbohydrate/protein/fat) were provided by Zone Labs, Inc. . Commercial gluten-free pasta  and commercial flaked cereal  were used as controls.2) were recruited from a campus community in the Phoenix metropolitan area via online advertisements and word of mouth in January 2015. Interested individuals were invited to complete an online survey to screen for history of food allergies or diet restrictions , insulin use, cigarette use, active disease states including diagnosed diabetes, anticipated changes to diet or physical activity levels, recent weight gain or loss (±10 pounds), and current or recent pregnancy or lactation. Prescription medication use by participants, including statins and hypertensive medications, was allowed if use had been consistent for the previous 3 months and would remain consistent during the trial. Volunteers were advised that the diet protocol was energy restricted to promote ~1 pound weight loss per week and that study foods  would be provided for participants as their primary foods for the duration of the trial. Qualifying individuals were invited to the study site to discuss the food trial in detail and provide written informed consent. The Institutional Review Board at Arizona State University approved the study. The study was registered at ClinicalTrials.gov (identifier: NCT02851498).Healthy adults by self-report  20–42 kg/mn = 49). Thirty-nine participants agreed to start the trial (January and February 2015) and were stratified by age, BMI, and TG/HDL ratio and randomly assigned by coin toss to one of two groups: Control  or high-protein . Participants were scheduled for weekly visits during the 6-week study to weigh-in and pick up food packages. CON and HPR test foods were similar in appearance and taste, and participants were blinded to the diet assignment. All participants received individual instruction on the diet protocol  and received personal diet plans for additional foods to be consumed in addition to the test foods. Individualized diet plans, designed to reduce total energy intake by 500 kcal below maintenance needs based on the Mifflin St Jeor equation, utilized an exchange system to promote healthy food choices for both groups in addition to the provided cereal and pasta dishes. Participants were counselled to eat low-fat dairy and meats as well as fruit and vegetables daily. The provided pasta meals followed a 7-day rotating menu. The participants received at no cost 14 different pasta dishes weekly during the study  along with seven prepackaged cereal aliquots. The fourteen pasta dishes were prepared weekly in the metabolic kitchen at Arizona State University and frozen prior to pick-up. The study foods provided 935 to 954 kcal on average each day. A fasting blood sample was collected from 92 consented adults  to determine the triglyceride/high-density lipoprotein (TG/HDL) ratio, an indicator of IR . Those aParticipants recorded study compliance  on a calendar and recorded food intake daily in a written journal. A fasting venous blood sample was collected at trial weeks 0 and 6, and body weight and composition was determined using a bioelectrical impedance scale  at these same time points using a standardized measurement protocol. Participants were asked to avoid moderate to heavy exercise on the day prior to blood collection and to fast overnight (no food or beverage with the exception of water for 10 h). The Profile of Mood States (POMS) questionnaire was also completed at trial weeks 0, and 6, and a physical activity questionnaire was completed at trial weeks 0, 3, and 6.Fasting blood samples were analyzed in duplicate for serum glucose and secondary outcomes  cholesterol, and high-sensitivity C-reactive protein [hsCRP]) using a point-of-care COBAS C111 chemistry random access autoanalyzer . Insulin concentrations were measured by radioimmunoassay , and HOMA-IR was calculated as (fasting insulin μU/mL × fasting glucose mg/dL)/405. Other secondary outcomes included total antioxidant capacity, measured using a commercially available kit according to the manufacturer’s instructions , and fasting plasma concentrations of thiobarbituric acid reactive substances (TBARS), measured using a kit  and quantitated as malondialdehyde which reacts with TBARS acid to form a product that is measured spectrophotometrically. In addition, glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were analyzed in an aliquot of blood that was immediately stabilized by the addition of aprotinin and the DPPV inhibitor, and GLP-1 and PYY were later measured by commercial kits according to manufacturers’ instructions (Millipore GLP-1 (Active) Cat# EGLP-35K and Millipore Human PYY  cat# EZHPYYT66K; EMD Millipore Corporation, Billerica, MA, USA). A chemiluminescence ELISA assay kit was used to quantitate high-molecular weight (HMW)-adiponectin . 8-isoprostane was measured using a competitive ELISA assay system .2p) controlling for confounding variables as indicated. Data were transformed to achieve normality prior to analyses if necessary. Analyses were performed using SPSS software, version 22 . A p-value < 0.05 was considered significant and a large effect size . Power calculations indicated that a total of 42 participants were necessary for this parallel-design study to detect a 2.0 unit difference in HOMA-IR , at an 8140; see ) was conn = 18) and those that did (n = 21). Moreover, using intention-to-treat calculations , the 6-week weight loss for all participants randomized to a treatment group did not differ significantly between groups . Hence, data are presented only for the 21 participants that completed the trial; the baseline data for these participants  are displayed in Of the 39 participants randomized to treatment groups , eight did not return to the test site after the baseline visit, and an additional 10 participants withdrew prior to the end of the 6-week trial. These latter participants withdrew from the trial during weeks 0–3 (with the exception of one that withdrew during week 4) due to time conflicts and/or a waning desire to consume the study test foods each day. No adverse effects were reported during the study. Baseline age, body weight, BMI, and the TG/HDL ratio did not differ significantly between those who did not complete the trial (p = 0.532). Both groups were advised not to change their daily exercise activity during the trial, and the physical activity (recorded as MET hours per week) did not differ between the CON and HPR groups at week 3 (43.8 ± 51.2 and 48.0 ± 33.4) or at week 6 (40.1 ± 21.7 and 46.6 ± 27.2).Study compliance (the self-reported consumption of study foods daily) was 81 ± 7% and 83 ± 13% for the CON and HPR groups respectively (n = 9 and n = 6 for the CON and HPR groups respectively) did not differ between groups, and the average energy restriction among participants ranged from 310 to 493 kcal/day for the CON and HPR groups respectively (p = 0.522). The final average macronutrient composition of the CON group was 51/15/33 for carbohydrate/protein/fat as per cent of total energy, and 42/23/36 for carbohydrate/protein/fat as per cent of total energy in the HPR group. The average combined fiber intake was similar in both groups (25 g per day in the CON group and 33 g per day in the HPR group). The 6-week mean weight loss was similar for the CON and HPR groups, −2.7 ± 2.6 and −3.2 ± 3.0 kg respectively (p = 0.801); however, the 6-week change in fat-free mass (FFM) differed significantly between groups with CON participants losing FFM while the HPR participants increased FFM  (p = 0.125 and 0.158).Diet record analyses indicated that participants adhered to their meal plans, and daily nutrient intakes from non-experimental foods did not vary by group . The ene= 0.344) . This pa= 0.344)  as well p = 0.020). This difference in HOMA-IR was primarily due to reductions in the fasting insulin concentrations that differed significantly between the two groups (p = 0.017). The change in HOMA-IR was inversely related to the change in FFM among participants . LDL cholesterol, triglycerides, total/HDL cholesterol, and TG/HDL cholesterol indices improved significantly over the 6-week trial among participants as a whole, but these indices did not differ between groups as a result of the intervention . The changes in the other blood indices were not related to weight change or to the change in FFM.The 6-week change in plasma glucose and insulin, HOMA-IR, GLP-1, PYY, HMW-adiponectin, and lipids by group are shown in rvention . The chap = 0.007); yet, the change in GLP-1 differed between groups during the trial . The changes in PYY and GLP-1 were correlated , and the change in PYY was related to the change in the TG/HDL ratio . Otherwise, there were no relationships between GLP-1, PYY, insulin sensitivity markers, or body composition, either at baseline or over the course of the trial. There were no differences between groups for blood markers of oxidative stress and inflammation  and the loss of body weight (2.7 to 3.2 kg) was modest for the 6-week trial reported herein; yet the reduction in HOMA-IR for the HPR group (−1.7) differed significantly from that for the control group (−0.7), and was greater than that noted for both the Tang et al. and Watson et al. reports. Interestingly, the protein content of the HPR diet herein was estimated at 1.4 g/kg, a level below that implemented by Tang et al. for their high-protein diet. Hence, the purposeful distribution of high-quality protein across meals  and the fact the protein was of a novel functional form may be important considerations when planning calorie-restricted diets to improve IR. Based on the work of Mamerow et al. , muscle There is little research available comparing the effect of dietary protein distribution on muscle mass in healthy, non-exercising adults. In hospitalized older adults, a population likely to experience sarcopenia, Bouillanne et al.  reportedParticipants who completed the current study were at heightened risk for metabolic syndrome based on their TG/HDL ratios, with one-third of participants displaying TG/HDL ratios above the cut-off for metabolic syndrome per group (≥3.5) . Unlike The possibility of performance bias due to non-blinding of participants is a limitation of weight loss trials comparing different macronutrient compositions ,17. UnleOther weight loss investigations also reported reductions in fasting PYY and GLP-1 concentrations, particularly in the early phase of energy restriction ,24,25, aMajor study limitations include the small sample size and short treatment duration; hence, to confirm these findings longer studies with sufficient numbers of participants are needed. FFM was estimated using bioelectrical impedance technology, which may overestimate FFM as compared to the gold standard, dual-energy X-ray absorptiometry. However, the differences in these techniques are proportional across measurements and likely related to the constants incorporated into the algorithms to estimate FFM using BIA . The attThis randomized controlled feeding trial demonstrated that a high protein diet  that include novel functional foods combined with modest calorie restriction was 140% more effective for reducing HOMA-IR in adults at risk for IR when compared to protein intake similar to a standard American diet  in the


E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussedFrataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on  It is involved in several central functions in cells, including delivery of iron to different biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase –5. Fratal matrix , 10. Therization , 11, 12.E. coli frataxin CyaY has been obtained [Bacilus subtilis has also been shown to interact with ferrochelatase, the terminal enzyme of heme synthesis that catalyzes iron insertion into protoporphyrin IX [Vibrio cholerae CyaY binds heme with dissociation constant in the nano-molar range [One of the primary functions of frataxin from bacteria, yeast and humans is related to its involvement in the ISC assembly machinery –18. A loobtained . CyaY frhyrin IX . In addiar range , and thaar range . In a laar range .The involvement of yeast frataxin Yfh1 in various cellular functions has also been demonstrated in a large number of studies. Thus, the iron storage function of Yfh1 was addressed by X-ray absorption spectroscopy (XAS) measurements of iron-loaded multimeric Yfh1, which showed that bound iron was in the ferric state, in the form of a ferrihydrite mineral, a biomineral composed of ferric oxide/hydroxide, which is also found in ferritin . SubsequUsing a combination of electron microscopy (EM) and SAXS, the structures of the oligomers formed by Yfh1 in the presence of iron could be resolved and were shown to be multimers of trimers , suggesting that a trimer is the smallest building block for the higher-order oligomers , 26, 27.42-210, FXN56-210, and FXN81-210. These isoforms are formed by the cleavage of the FXN precursor by the mitochondrial processing peptidase after the transfer of the protein to the mitochondrial matrix [42-210 and FXN56-210, have been shown to oligomerize independently of iron during expression in E. coli [56-210 was also found to store iron and to build a ferrihydrite mineral core by catalyzing iron oxidation through the ferroxidation reaction [42-210 into large particles was recently revealed by EM single particle reconstruction of the complex of the 24-meric FXN42-210 particle in complex with the ISC synthesis machinery proteins NFS1:ISD11:ISCU [56-210 obtained after dissociation of the oligomers, could be re-assembled into oligomers after incubation at moderate concentrations (1.5 M) of GdmCl and subsequent dialysis of the denaturant [In contrast to bacterial and yeast frataxin, human frataxin (FXN) has been found to exist in three different isoforms: FXNl matrix –31. The  E. coli , 32. Simreaction . The pacD11:ISCU . Also inD11:ISCU , 34. Mor81-210, which due to the absence of a long N-terminal extension may have properties comparable to those of CyaY, is the most abundant form of human frataxin in mitochondria. It is believed to exist predominantly in a monomeric state and to have labile iron binding capacity [81-210 may form oligomers in vitro at aerobic conditions in the presence of iron and that the addition of ferric iron chelators deferiprone and DFO stabilizes these oligomers and even stimulates the formation of a larger number of oligomers [81-210 oligomers were unstable and were found to dissociate after approximately 24 hours [The shortest isoform of human frataxin, FXNcapacity , 35, 36.ligomers . Interes24 hours . CyaY ol24 hours .Although frataxin has a highly conserved amino acid sequence and three-dimensional structure , the datE. coli CyaY was studied using a combination of methods, which include size-exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), electron microscopy (EM), native mass spectroscopy (native MS) and nano-differential scanning fluorimetry (nano-DSF). The study addresses the issues of CyaY oligomer formation and the possible architecture and stability of the oligomers.Here, iron-induced oligomerization of 280 nm) of 28,990 M-1 cm-1. The protein was eluted in HN100 buffer .CyaY was purified as described in , with th4)2Fe(SO4)2.6H2O) at different Fe2+ concentrations that varied from 0.04 mM to 0.82 mM . The incubation was performed at 30°C for one hour according to published protocols [For studying iron-induced oligomerization, CyaY at a concentration of 1 mg/ml (0.082 mM) in HN100 buffer was incubated with ammonium iron (II) sulphate hexahydrate  = 1.632.10-2cm-1, at 20°C),) and water measurements (empty capillary for subtraction) we could set our data to absolute scale in order to make molecular weight estimations for the samples [For the time-resolved SAXS (TR-SAXS) data, a sample with a 7:1 Fe, France . Using t samples . Bovine  samples . Scatter(0) and Rg determinations were made using the Guinier approximation. GNOM [g, Porod volume Vp, and the maximum particle dimension Dmax derived from the pair distribution function p(r).Data processing for all SAXS samples was achieved using PRIMUS software  and the on. GNOM  was usedhead-to-tail and head-to-head arrangements). The fourth model was a tetramer generated earlier based on a Yfh1 hexamer and used for modeling CyaY SAXS oligomer data [The OLIGOMER program  was usedmer data . CRYSOL mer data  was used4)2Fe(SO4)2.6H2O) at the molar ratio of 7:1 iron to CyaY at 30°C for 1 h before loading on Superdex 75 10/300 GL columns . The final concentration for CyaY was 8 mg/ml (0.65 mM) with 4.55 mM Fe2+ (a 7:1 ratio of iron to protein). Since the 7:1 iron-to-protein ratio resulted in protein aggregation at protein concentrations higher than 3 mg/ml, 100 μl aliquots of 2 mg/ml CyaY were initially incubated with iron and subsequently concentrated to the required concentration. The sample was always centrifuged for 10 min at 14000 rpm before loading on the columns. The columns were pre-equilibrated with HN100 buffer and operated at a flow rate of 0.5 ml/min at room temperature. Sample elution followed at 280 nm. The size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) experiment was optimized to obtain a well-separated peak for the iron-induced oligomeric species. The best conditions for the experiment included an iron incubation time of 45 min, followed by filtering the sample using a 100 kDa cut-off membrane (Amicon ® Ultra) to remove aggregates and larger oligomeric particles (> 100 kDa) that could not be separated by the column. The flow-through was then concentrated in a 10 kDa cut-off membrane (Amicon ® Ultra) to 100 μl before loading onto the SEC column.CyaY was incubated with ammonium iron (II) sulphate  at a 7:1 iron-to-protein ratio were prepared as described above for SAXS experiments. The sample was diluted to 0.4 mg/ml and applied to a 400-mesh carbon copper grid (EMS). The grid was pre-incubated in HN100 for 1 minute before applying the sample. The sample buffer was blotted and washed for 3 seconds in sterile water. Uranyl acetate (1% w/v) was applied to the grid for 30 seconds, and excess stain was blotted directly. The grid was left to dry for 30 minutes before insertion into the sample holder of a Philips CM120 transmission electron microscope equipped with a GATAN GIF 100 energy filter and a GATAN 791 CCD camera (1024x1024 pixels). All images were taken at 55000x magnification. Glow discharge was applied for more uniform spreading of protein sample on the grids.2+-to-CyaY ratios and buffer used in the SAXS experiments . The iron-incubated samples had a protein concentration of 1 mg/ml and were prepared in the same way as for the SAXS experiments. Ten μL of each sample was loaded into UV capillaries (NanoTemper Technologies). The temperature gradient was set at 0.5°C/min in a range from 20 to 95°C. Protein unfolding was detected by following the change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. The ratio between the emission intensities at 350 nm and 330 nm (F350/F330) was used to track the structural changes with increasing temperature. Data analysis was performed using the manufacturer’s software, where Tm was calculated using the peaks in the first derivative curve.Nano-DSF measurements were performed using a Prometheus NT.48 instrument . The measurements were done for CyaY without added iron and with the same FeAll samples were analyzed by using the SYNAPT G2-Si High-Definition Mass Spectrometer  , 52. For2+ in solution, Fe3+ is essential for oligomer formation and stabilization. These results were confirmed in our earlier work [g, maximum particle dimension Dmax, and Porod volume Vp) calculated in the absence of added iron and after a gradual increase of the iron-to-protein ratio. In the absence of iron, the data did not show any protein concentration dependence effects  and yielded an Rg of 1.54 ± 0.01 nm and a Dmax of 5.29 nm. This value is close to that observed earlier (1.38 ± 0.03 nm) [g was found to be due to using two different versions of PRIMUS [g and Dmax values were similar to those obtained in this study. In addition, for iron-incubated samples, differences in Rg may result from slight differences in sample preparation, such as small pipetting errors, since very low volumes of iron (between 0.5 to 1 μl) were added to the sample. Finally, variations in the uncontrolled iron oxidation in solution may also affect the results, as the experiments were run aerobically. The estimated Vp for apo CyaY was 21.24 nm3, which corresponds to an estimated molecular weight of 13.28 kDa. This value is within the normal range of the 10% error for SAXS measurements [Earlier studies of iron binding to CyaY showed that it could bind at least two ferrous and up to 25–26 ferric ions/monomer [ier work . Here, i0.03 nm) . The slif PRIMUS . When thurements , as the g, Dmax, and Vp , indicating that iron oxidation and the concomitant oligomerization were complete after this period. As noted above, earlier studies showed that after approximately 50 min of incubation at atmospheric oxygen, absorbance of oxo/hydroxo Fe(III) species measured at 305 nm reaches maximum [For the time-resolved study, we followed the course of CyaY oligomerization for one hour at 30°C. The iron-to-protein ratio of 7:1 was chosen to make sure that no protein aggregation occurred in the sample, and using protein concentration of 3 mg/ml. Feme point . The fig monomer . Since, ructures . At 80 mNo detailed experimental structural information on monomer packing within CyaY oligomers is available. A tetramer that was generated by analyzing the conserved interactions in Yfh1 hexamers was used earlier for fitting SAXS data and was suggested to be the building block of higher-order oligomers . Here, wg was found to be 2.37 ± 0.01 nm, and Dmax was 8.29 nm. The Vp was 36.02 nm3, which corresponds to a calculated molecular weight of 22.5 kDa. Taking into account an approximate 10% error in the accuracy of the molecular weight estimation from SAXS measurements [ab initio shape reconstruction program was used to generate models for CyaY dimers. The 20 output models had a mean value for the NSD of 0.658, which is within the normally accepted range for this parameter.The results of the SEC-SAXS experiments are summarized in urements , the obthead-to-tail arrangement of monomers, in which the N-terminus (residues 1–3) and the connecting loop between strands β3 and β4 (residues 54–58) of one monomer faced the connecting loop between helix 1 and strand β1 (residues 22–30) and the loop between strands β2 and β3 (residues 43–46) of the second monomer. The second model  faced the connecting loop between strand β3 and β4 (residues 56–59) of the second monomer. The N-terminus of the other subunit faced strand β5 (residues 68–74) of the neighboring monomer. It should be noted that cross-linking excluded a tail-to-tail arrangement of the monomers. The SASREF fitting of the first model (head-to-tail)  was also good, with χ2 = 0.72.SASREF  was usednd model  had a heto-tail)  was veryhead-to-tail arrangement is also supported by the observation that the residues involved in metal binding [ab initio model onto the head-to-tail dimer generated by SASREF  . The  of 1.12 . Using t = 0.88) . Earlierg, Dmax, and Vp values for the mixture identified by SAXS were the highest, suggesting the most abundant content of oligomeric species. The native MS analysis showed a clear signal for the monomer, a weak signal for the dimer, and a yet weaker signal for the trimer . For comparison, the protein in the absence of iron was also measured. The melting point (Tm) for each sample was obtained from the plot of inflection points in the first derivative curve of the emission intensities ratio at 350 nm and 330 nm (F350/330) against the temperature . At higher ratios, 4:1, 7:1, and 10:1, there were two inflection points at which two different Tm values could be identified—the first was between 64.4°C and 65°C, similar to the values observed for protein without iron and at low iron-to-protein ratios, and the second was substantially lower, ranging between 40°C and 41.5°C. This Tm presumably corresponds to dissociation of higher-order oligomeric species.CyaY oligomers have been shown to have higher stability than oligomers of the short variant of human frataxin FXNter 24 h . Here, wperature . The resperature  show thaE. coli CyaY. Using SEC-SAXS and native MS we show that in the presence of iron a dimer is one of the most common multimeric forms of CyaY, although higher order oligomers like trimers, tetramers, pentamers, and perhaps even hexamers may exist in solution. The EM data together with earlier DLS data [In this work we continue our explor0ation of the details of iron-induced oligomerization of frataxin and focus on DLS data , show thDLS data –57. In aDLS data . The tetDLS data ), fits t3+ hydroxo(oxo) mineral [Both the SAXS and EM data show that higher iron concentrations lead to the formation of larger oligomeric species and that oligomerization proceeds in a linear fashion and is directly dependent on iron oxidation. Earlier it was shown that CyaY binds ferrous iron at the stoichiometry of two iron ions/monomer, while ferric iron may bind at a stoichiometry of up to 25 ions/monomer, which are stored as a polynuclear Fe mineral , 58, it  mineral ), sugges mineral . The sliThe dimer model of CyaY in the presence of iron was deduced from our current SAXS data, but it also agrees with the earlier suggested packing of monomers, which was based on cross-linking mass spectrometry and docking studies and that also suggested a head-to-tail arrangement . This ar81-210 oligomers, as compared to CyaY oligomers [Residues D3, E19, D23 D55 and H58 have been found to interact with metals in crystals . The aciligomers .E. coli CyaY sequences, respectively) to the first β-strand of the central β-sheet . In the human protein, for example, there are in total 13 acidic residues in the cluster, 8 of which are located in helix 1. In yeast and E. coli 5 and 7 acidic residues  are found in helix 1. However, only 3 of the cluster residues have conserved position across species (Burkholderia cenocepacia (PDB entry: 4JPD) has only 5 acidic residues in helix 1, which is similar to the yeast structure. Analysis of the packing of the molecules within the crystals of B. cenocepacia CyaY shows that the monomers are arranged as two trimers packed against each other around a threefold symmetry axis   , while Eructures . Further81-210 [Due to the low signal/noise ratio of the higher oligomeric species, it was difficult to detect their presence reliably in native MS and SEC-SAXS studies. Only monomers, dimers, and trimers could be reliably detected. The low volume fractions of the larger oligomeric species may be a result of lower stability, as shown by nano-DSF. The presumed dissociation of oligomers may also be a result of the buffer conditions used, which included acetate, a compound known to chelate iron. Further optimization of the experiments may be required in this case. However, the data clearly demonstrated that native MS can be used in the study of this system. It should be noted that the trimers observed in native MS could also be identified using the SDS-PAGE of crosslinked samples of CyaY and FXN81-210 .in vitro results, specially when protein-protein interactions and protein oligomerization are concerned, are not always easy to verify directly in living cells, it still should be possible to link the findings to processes observed in vivo. As noted in the introduction, one of the primary functions of CyaY is related to its involvement in the ISC assembly machinery [Although achinery , 61, butachinery , and preachinery . In addiS1 FigFor the analysis, a 7:1 iron-to-CyaY ratio sample was used. Numbers indicate detected ionization states of CyaY.(PDF)Click here for additional data file.


We aimed to evaluate the comparative efficacy and safety of dipeptidyl peptidase-4 inhibitors (DPP4i), glucagon-like peptide-1 receptor agonists (GLP-1RA), sodium-glucose co-transporter 2 inhibitors (SGLT2i), or thiazolidinedione (TZD) as an adjunctive treatment in patients with poorly controlled type 2 diabetes mellitus (T2DM) on insulin therapy. We searched Medline, Embase, the Cochrane Library, and ClinicalTrials.gov through April 2016. Bayesian network meta-analyses were performed with covariate adjustment. The primary outcome was the change in glycated hemoglobin A1c (HbA1c) from baseline. Fifty randomized controlled trials covering 15,494 patients were included. GLP-1RA showed the greatest HbA1c-lowering effect compared to the control , followed by TZD , SGLT2i , and DPP4i . SGLT2i showed the greatest fasting plasma glucose reduction. GLP-1RA and SGLT2i showed greater body weight reduction, whereas TZD increased body weight. TZD was ranked the highest in terms of insulin dose reduction. The risk of hypoglycemia was increased with TZD or GLP-1RA. The study provides the best available evidence on the comparative efficacy and safety of non-insulin anti-diabetic agents on top of pre-existing insulin therapy for inadequately controlled T2DM patients. Therefore, as an alternative to more intensive insulin therapy, a non-insulin anti-diabetic drug may be used as an add-on drug to insulin therapy. In this regard, recent guidelines for T2DM management have recommended the use of dipeptidyl peptidase-4 inhibitors (DPP4i), glucagon-like peptide-1 receptor agonists (GLP-1RA), sodium-glucose co-transporter 2 inhibitors (SGLT2i), or thiazolidinedione (TZD) on top of pre-existing insulin therapy, particularly basal insulin4. Each agent has both advantages and disadvantages according to the characteristics of the drug, which need to be considered when determining the most appropriate treatment for an individual patient. To the best of our knowledge, there has been no comparative efficacy or effectiveness study of adjunctive treatments to insulin therapy. Hence, we performed a systematic review with a network meta-analysis to evaluate the comparative efficacy and safety of DPP4i, GLP-1RA, SGLT2i, and TZD as an adjunctive treatment in patients with poorly controlled T2DM on insulin therapy.Impaired insulin secretion in the presence of insulin resistance is the key feature of type 2 diabetes mellitus (T2DM). The progressive nature of insulin secretory failure makes T2DM patients require insulin therapy to achieve their glycemic goalsWe conducted a systematic review and network meta-analysis following a pre-developed protocol (Supplementary Appendix 1).The following electronic databases were searched from inception to April 2016: Medline, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), and ClinicalTrials.gov. The detailed search strategy used in Medline is available in Supplementary Appendix 2.We included randomized controlled trials (RCTs) that investigated the effect of DPP4i, GLP-1RA, SGLT2i, or TZD as an add-on drug to pre-existing insulin therapy in patients with T2DM. Both placebo-controlled and open-label trials were included, and concurrent use of other anti-diabetic agents was allowed. Studies reporting the change of HbA1c from baseline were included when some information on the mean and its variability was available. An intervention of at least 12 weeks was required and studies in English were considered for inclusion. Duplicate publications or studies on post-hoc analysis were excluded. We also excluded studies with patients treated using an insulin pump. Two reviewers (J.-H.Y. and S.H.M.) independently screened all studies by title and abstract, and then by full text, to assess the eligibility of the studies. Any discrepancies between the authors were resolved through mutual discussions with the other authors .We extracted the following information from each study: study information, participant characteristics at baseline, information on interventions and values of outcome variables . We attempted to classify various insulin regimens into 2 categories: a stable insulin dose group, in which the insulin doses were kept relatively constant throughout the study duration unless the dose needed to be altered for safety reasons, and an insulin dose titration group, in which the insulin doses were titrated according to study-specific predefined titration algorithms. We obtained information from the ClinicalTrials.gov website when the studies had never been published as an article.5, and any controversies were resolved by mutual discussion. We considered the 6 aspects of risk of bias, including the adequacy of random sequence generation, allocation concealment, blinding of participants and personnel, completeness of outcome data, selective reporting, and other sources of bias. For the completeness of outcome data, analyses based on the intention-to-treat principle or the full analysis set were considered to be low-risk. Selective reporting was assessed as high-risk when any results were regarded as missing while some outcome variables or conditions of analysis were mentioned in the articles or when the publication was not considered as in full. Other sources of bias were assessed according to the comparability of the baseline characteristics of the participants in the trial groups.Two independent reviewers (J.-H.Y. and S.H.M.) assessed the study quality and risk of bias according to the Cochrane Collaboration tool7. This was done primarily by a qualitative review of the regimen used in each study, and by a quantitative comparison of the results from the control group in all included studies using the 2 test and I2 statistics, with a forest plot presenting the pooled weighted mean and the 95% confidence intervals (CIs). We considered that participants’ age, sex, body mass index (BMI), baseline HbA1c, duration of diabetes, and baseline insulin dose could affect within-group or between-group heterogeneity, and therefore could be potential confounders. Although we reviewed each insulin titration algorithm used in all the included studies, it was not possible to include it as a covariate for adjustment due to the diversity of algorithms. However, since the treatment results from the control group inherently reflect the effects of various insulin regimens, we used them as a modifier representing unmeasured prognostic factors related to various insulin regimens across the included trials8. We explored the potential relationship between each candidate covariate and the outcome by a scatter plot with a conventional random-effects meta-regression. The network meta-analysis was conducted using a Bayesian approach, and the posterior distribution for each parameter of interest was summarized with a median and the 95% credible interval (CrI)10. We selected covariates that contributed to the network meta-analysis, including each as a covariate at a time, based on the 95% CrI of the coefficient. Covariates with a strong clinical rationale were also selected. The final network meta-analysis was adjusted for the selected covariates with a random-effects model. We performed sensitivity analyses after mean imputation of the values of covariates missing in some studies, and additionally by including only placebo-controlled trials. We estimated the surface under the cumulative ranking curve11 for each treatment for each outcome, which provides the probability for each treatment to be ranked as the most effective treatment or to have the highest chance for a respective adverse event. We used STATA version 12 , R version 3.2.1 , and WinBUGS version 14 .For the continuous variables, pooled weighted mean differences between treatment groups were calculated. For the dichotomous variables, we calculated the pooled relative risks (RR). Prior to conducting the network meta-analysis, homogeneity in the common comparator  was assessed as an indicator of study comparabilityThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.55. We additionally selected 6 trials61 among the 1092 clinical trials identified from ClinicalTrials.gov, of which 3 trials59 were unpublished. The network frame based on the 50 studies is depicted in Fig. 61, 13 GLP-1RA59, 9 SGLT2i60 and 13 TZD54 studies included in the network. No study with head-to-head comparisons between the non-insulin agents was found. The characteristics of the included trials are described in Table 61 were considered as having a stable insulin dose, whereas 19 studies56 involved insulin dose titration. The rest59 of the studies could not be classified in either group because they did not describe the method of insulin dosage. Two studies56 with fixed ratio combination products of GLP-1RA and insulin were included.The flowchart of the literature selection is shown in Fig. 61 (54%) and twenty-three61 (46%) of the 50 studies did not describe their methods of generating a random number and of allocation concealment, respectively. In the assessment for the blinding of patients and personnel, 8 trials56 (16%) were evaluated as high-risk, as they were open-label trials. For the risk of incomplete outcome data, most studies61 (80%) were rated as low-risk. Three studies59 were considered open to a high risk of selective reporting bias since we used their data from the Clinicaltrials.gov website only, and those data were not regarded as complete reports. Most of the trials61 (90%) had balanced baseline characteristics between the treatment groups. The detailed results of the assessment are found in Supplementary Figure Twenty-seven2 = 94.5%, 97.9%, 94.7%, and 80.5%, respectively) . Greater reduction of HbA1c in the control group was noted in some studies with GLP-1RA or TZD treatments and those also adopted some type of insulin titration method. An inherited relationship was observed between the change in the HbA1c in the control group and the treatment difference in the changes of the HbA1c (P = 0.02)  , −0.55% (−0.82% to −0.27%), −0.05% (−0.21% to 0.10%), and −0.11% (−0.32% to 0.09%) in the DPP4i, GLP-1RA, SGLT2i, and TZD studies, respectively, with a large extent of within-group heterogeneity ; GLP-1RA, −0.84% (−1.00% to −0.69%); SGLT2i, −0.66% (−0.84% to −0.48%); and TZD, −0.73% (−0.93% to −0.52%)  [−0.63 mmol/L (−0.86 to −0.41 mmol/L)]; GLP1-RA, −9.96 mg/dL (−14.55 to −5.38 mg/dL) [−0.55 mmol/L (−0.81 to −0.30 mmol/L)]; SGLT2i, −24.14 mg/dL (−29.64 to −18.54 mg/dL) [−1.34 mmol/L (−1.65 to −1.03 mmol/L)]; TZD, −19.57 mg/dL (−24.78 to −14.25 mg/dL) [−1.09 mmol/L (−1.38 to −0.79 mmol/L)] , whereas TZD showed an increase in body weight compared to DPP4i     GLP-1RA showed the greatest effect on HbA1c reduction, followed by TZD, SGLT2i, and DPP4i; (2) the reduction in FPG was higher with SGLT2i than with DPP4i and GLP1-RA; (3) GLP-1RA and SGLT2i were associated with body weight reduction, whereas TZD increased body weight; (4) TZD and GLP-1RA reduced total daily insulin requirements; (5) the risk of hypoglycemia was increased with TZD and GLP-1RA.62.The studies included in our analysis had different baseline characteristics and showed significant within-group and between-group heterogeneity. We found that age, sex, and BMI at baseline were associated with placebo-subtracted HbA1c reduction in a linear relationship; therefore, we adjusted for those potential confounding factors as covariates. In addition, since it was not possible to adjust for various methods of insulin titration among the included studies, we instead adjusted for the treatment effects of the control group. After adjustment for the covariates, GLP-1RA was at the top rank of the hierarchy. In addition, the results were similar in the sensitivity analyses, with GLP-1RA showing the highest probability of being the best for glycemic control. Consistent with the primary outcome, GLP-1RA also showed the highest rank for achieving HbA1c levels <7.0%. This might be explained by the complementary effects of GLP-1RA with insulin therapy63. In contrast, GLP-1RA agents have different FPG- and PPG-lowering efficacy, depending on their duration of action. Short-acting GLP-1RA drugs drastically decelerate gastric emptying, leading to a marked reduction in the PPG excursion. In contrast, long-acting GLP-1RA drugs have little effect on gastric emptying, but their long-lasting effect on pancreatic beta-cell and alpha-cell function contributes to the predominant reduction in FPG levels64. Because about half of the GLP-1RA studies used long-acting GLP-1RA agents, and the rest of the studies used short-acting GLP-1RA agents, the FPG-lowering effect of add-on GLP-1RA might be mixed.The FPG-lowering efficacy of SGLT2i was significantly greater than that of DPP4i and GLP-1RA, and tended to be greater than that of TZD. SGLT2i drugs act on the kidneys and increase glucose excretion through the urine, which is mediated by an insulin-independent mechanism; therefore, they reduce both FPG and postprandial glucose (PPG)65. In this network meta-analysis, GLP-1RA appeared to be the most effective for weight reduction, which is attributed to decreasing appetite and promoting satiety64. SGLT2i was ranked lower than GLP-1RA, possibly because the weight-loss effect of SGLT2i is compensated by increased appetite and calorie intake67. Overall, the reported effects of weight loss with GLP-1RA and SGLT2i, weight gain with TZD, and no effect for DPP4i were preserved when combined with preexisting insulin therapy in patients with T2DM68.Weight gain is commonly associated with insulin therapy, particularly with intensive insulin therapy69. Differences in the insulin titration algorithm may also have contributed to the different insulin-sparing effect across the studies. More than half of the studies of TZD and GLP-1RA adopted insulin dose titration regimens, whereas most studies of SGLT2i and DPP4i adopted stable insulin dose regimens. The placebo-subtracted insulin-sparing effects in studies with active insulin dose titration tend to be greater than in studies with stable insulin dose regimens.The insulin-sparing effect, which was pre-defined as the reduction in the total required daily dose of insulin from baseline, was greater in TZD and GLP-1RA. The greater potency of GLP-1RA and TZD in glycemic control and their insulin-sensitizing effects may contribute to reducing insulin dosage71. DPP4i drugs also have a minimal risk of hypoglycemia due to their glucose-dependent mechanism of action regarding the regulation of insulin and glucagon secretion62. Theoretically, TZD and GLP-1RA drugs seldom provoke hypoglycemia by themselves unless combined with insulinotrophic agents72. In this study, however, TZD and GLP-1RA agents showed an increased risk of hypoglycemia in comparison to the control group. The greater glucose-lowering potency of TZD and GLP-1RA and adopting an active insulin dose titration regimen might contribute to the increased risk. Because the definitions of hypoglycemia varied widely across the included studies, the comparative risk of hypoglycemia among these agents must be carefully interpreted.In the current study, SGLT2i and DPP4i were suggested to have a potentially lower risk of hypoglycemia than the other 2 agents. In general, SGLT2i drugs do not induce hypoglycemia because they increase plasma glucagon concentrations and decrease plasma insulin concentrationsThe study has several limitations. First, the current study is based on indirect comparisons using a common control. Network meta-analyses are typically required to address inconsistencies between the results of direct and indirect comparisons. However, since no study has conducted head-to-head comparisons among GLP-1RA, DPP4i, SGLT2i, and TZD, it was not possible to evaluate consistency between the results of direct and indirect comparisons. We therefore primarily explored the heterogeneity among the values of HbA1c reduction in the control group, which possibly indicates a lack of comparability among the included studies. We then identified as many potential confounders as possible and adjusted for their effect in the final analysis. Second, the patients in the studies included in this study used various baseline insulin regimens, but we could not isolate the effects of the studied medications on top of the basal insulin therapy regimen, which is the most common practice of insulin therapy at present. Finally, due to lack of information, we could not assess drug-specific safety outcomes, such as gastrointestinal adverse events and urinary tract infections.56. For patients who need to lower their FPG level, SGLT2i might be a good treatment option due to its superior FPG-lowering efficacy without increasing weight gain or hypoglycemia. It also has the advantage of body weight reduction. Even though DPP4i was relatively less efficacious for HbA1c reduction, it rarely increased the risk of hypoglycemia. Therefore, DPP4i may be considered for patients with relatively less severe hyperglycemia but with a high risk of hypoglycemia. Lastly, TZD might be taken into consideration in patients with severe insulin resistance requiring high doses of insulin.Based on the evidence from our network meta-analysis, we may suggest patient-centered guidance for non-insulin anti-diabetic agents in patients with inadequately controlled T2DM on pre-existing insulin therapy. For those who need to reduce both HbA1c and body weight, GLP-1RA might be an optimal choice. Fixed-ratio combinations of insulin and GLP-1RA are currently available to improve glycemic control and to minimize the risk of weight gain73, our systematic review and network meta-analysis provides evidence-based suggestions for selecting an appropriate non-insulin antidiabetic agent based on patients’ clinical characteristics. Nonetheless, RCTs directly comparing the efficacy and safety of these agents should be undertaken.Since a previous study which compared insulin monotherapy with the addition of various oral glucose-lowering agents to insulin did not present the comparative efficacy among non-insulin anti-diabetic agents in patients with inadequately controlled T2DM with insulin therapySupplementary informationPRISMA NMA checklist


When people hear unexpected perturbations in auditory feedback, they produce rapid compensatory adjustments of their vocal behavior. Recent evidence has shown enhanced vocal compensations and cortical event-related potentials (ERPs) in response to attended pitch feedback perturbations, suggesting that this reflex-like behavior is influenced by selective attention. Less is known, however, about auditory-motor integration for voice control during divided attention. The present cross-modal study investigated the behavioral and ERP correlates of auditory feedback control of vocal pitch production during divided attention. During the production of sustained vowels, 32 young adults were instructed to simultaneously attend to both pitch feedback perturbations they heard and flashing red lights they saw. The presentation rate of the visual stimuli was varied to produce a low, intermediate, and high attentional load. The behavioral results showed that the low-load condition elicited significantly smaller vocal compensations for pitch perturbations than the intermediate-load and high-load conditions. As well, the cortical processing of vocal pitch feedback was also modulated as a function of divided attention. When compared to the low-load and intermediate-load conditions, the high-load condition elicited significantly larger N1 responses and smaller P2 responses to pitch perturbations. These findings provide the first neurobehavioral evidence that divided attention can modulate auditory feedback control of vocal pitch production. Auditory feedback is critical for the production of proper speech sounds : 21.53 ± 2.41 years] entered the final statistical analyses. They were all right-handed, had normal or corrected-normal vision, and reported no history of hearing, speech, language, or neurological disorders. Hearing thresholds were screened at 25 dB HL for octave intervals of 500–4000 Hz. Written informed consent was obtained from all participants. The research protocol that was in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) was approved by the Institutional Review Board of The First Affiliated Hospital at Sun Yat-sen University of China.The experiment was conducted in a sound-attenuated booth, where participants' voice and electroencephalographic (EEG) signals were recorded. In order to partially mask the air-born and bone-conducted feedback, we calibrated the acoustic recording system so that the intensity of voice feedback the participant heard was 10 dB SPL higher than that of his/her voice output. During the experiment, participants' voice signals were transduced by a dynamic microphone  and sent to an Eventide Eclipse Harmonizer via a MOTU Ultralite Mk3 Firewire audio interface. A custom-developed Max/MSP software program (v.5.0 by Cycling 74) controlled the Harmonizer to pitch-shift the voice signals and sent them to an ICON NeoAmp headphone amplifier. The amplified pitch-shifted voices were presented to participants as attended auditory stimuli through insert earphones . Two circles representing the blue and red indicator lights were generated by the Max/MSP software program and displayed on the computer screen. The blue indicator light was used to cue the start and end of vocalization, while the flashing red indicator lights were used as attended visual stimuli. Transistor-transistor logic (TTL) control pulses were also generated by this program to mark the onset of the pitch perturbations. The original and pitch-shifted voice signals as well as the TTL control pulses were sampled at 10 kHz by a PowerLab A/D converter  and recorded using LabChart software (v.7.0 by AD Instruments).in≈200 MΩ; Electrical Geodesics Inc.), digitized at 1 kHz, and recorded using NetStation software . The TTL control pulses that signaled the onset of the pitch perturbations were sent to the EEG recording system via a DIN synch cable. The EEG signals across all channels were referenced to the vertex (Cz) during the online recording. Electrode impedances were maintained at ≤50 kΩ for individual sensors . Scalp-recorded brain potentials were amplified by a Net Amps 300 amplifier that accepts scalp-electrode impedances up to 40–60 kΩ  while seeing a number of red indicator light flashes on the computer screen. The number of the pitch perturbations ranged from one to five per vocalization. The first pitch perturbation occurred 500–1000 ms after the onset of vocalization, and the succeeding stimuli were presented with an inter-stimulus ISI of 700–900 ms. The red indicator light flashed 1–13 times per vocalization. The first red indicator light began to flash 500 ms after the blue indicator light prompted participants to vocalize, and the succeeding stimuli were presented with three different ISIs: 1400–2000 ms , 900–1500 ms , and 400–1000 ms . The onsets of auditory and visual stimuli were asynchronous. The durations of both the red indicator light and pitch perturbation were fixed at 200 ms. Production of ~40 consecutive vocalizations constituted one block, which led to ~100 trials  per condition. The order of the three attentional load conditions was counterbalanced across all subjects.While producing sustained vocalizations, participants were required to divide their attention to auditory (pitch feedback perturbations) and visual stimuli (flashing red lights) across the three load conditions. An immediate recall test was performed after each vocalization, during which they reported the number of the pitch perturbations that they heard and the number of the red indicator light flashes that they saw. This test ensured that participants attended to the bimodal stimuli as required. Their behavioral performance, as indexed by the percentage of correctly remembered auditory and visual stimuli across the three load conditions, was evaluated and submitted to statistical analyses.0 contours in Hertz were extracted from the voice signals using Praat software ) [reference = 195.997 Hz (G3)]. The voice contours in cents were then segmented into epochs of 200 ms before to 700 ms after the onset of the pitch perturbation. All individual trials were carefully inspected using a waterfall procedure and trials with signal processing errors or unexpected vocal stops were rejected from further analyses. Finally, the artifact-free trials were normalized by subtracting the mean F0 values in the baseline period (−200 to 0 ms) from the F0 values after the perturbation onset and then averaged to generate an overall response. The magnitude of a vocal response in cents was measured as the greatest F0 value following the response onset. The latency was defined as the time when the voice F0 contours exceeded 2 SDs above or below the pre-stimulus mean following the perturbation onset.The event-related averaging technique  to pitch feedback perturbations were analyzed using repeated-measures analysis of variance (RM-ANOVAs) in SPSS (v. 16.0). The magnitudes and latencies of vocal responses were subjected to one-way RM-ANOVAs, in which attentional load  was chosen as a within-subject factor. The amplitudes and latencies of N1 and P2 responses from 10 fronto-central electrodes  were subjected to three-way RM-ANOVAs, including three within-subject factors of attentional load, anteriority, and laterality. Frontal  and central  electrodes were chosen as an anteriority factor, while lateral left , medial left , midline , medial right , and lateral right  were used as a laterality factor. The Greenhouse-Geisser was used to correct probability values for multiple degrees of freedom when the assumption of sphericity was violated. Effect size was calculated using partial ηt(31) = 12.852, p < 0.001] and low-load conditions (98.0 ± 0.4%) . Also, their response accuracy in the intermediate-load condition was also significantly lower than accuracy in the low-load condition . These results indicate that poorer behavioral performance was associated with faster presentation rate of the red indicator light.Figure t(31) = 16.880, p < 0.001] and low-load conditions (96.4 ± 0.5%) . Their response accuracy in the intermediate-load condition was also significantly lower than that in the low-load condition . Therefore, response accuracy for identification of the number of the pitch perturbations was modulated by attentional load created by the different presentation rates of the red indicator lights that participants had to simultaneously count.Likewise, participants' accuracy for identifying the number of the pitch perturbations in the high-load condition (65.7 ± 1.1%) was significantly lower than both the intermediate-load (80.6 ± 0.9%) . Post-hoc Bonferroni comparison tests showed that the low-load condition (16.5 ± 1.8 cents) elicited significantly smaller response magnitudes than the intermediate-load (20.2 ± 2.4 cents) (p = 0.029) and high-load conditions (23.5 ± 4.0 cents) (p = 0.015) . In contrast, the latencies of vocal responses did not vary as a function of attentional load  .Figure F = 8.744, p = 0.001, partial η2 = 0.215]. Post-hoc Bonferroni comparison tests showed significantly larger N1 amplitudes (more negative) in the high-load condition relative to the intermediate-load (p = 0.009) and low-load conditions (p = 0.002) . Larger N1 amplitudes for the frontal electrodes relative to the central electrodes led to a significant main effect of anteriority . There was a significant main effect of laterality , which was caused by smaller N1 amplitudes for the left medial electrodes relative to the left lateral (p < 0.001) and central electrodes (p < 0.001).Figure F = 1.426, p = 0.248, partial η2 = 0.044] and anteriority  did not reach significance, whereas a significant main effect of laterality was observed . Post-hoc Bonferroni comparison tests showed significantly longer N1 latencies for the right lateral electrodes relative to the medial left (p = 0.013), medial right (p = 0.009), and middle electrodes (p = 0.001).For the N1 latencies, the main effects of attentional load . Post-hoc Bonferroni comparison tests showed that P2 amplitude was smaller in the high-load condition as compared to the intermediate-load condition (p < 0.001) and low-load condition (p < 0.001), and P2 amplitude was also smaller in the intermediate-load condition than the low-load condition (p < 0.001)  = 46.923, p < 0.001, partial η2 = 0.602] reached significance, as reflected by significantly smaller P2 responses for the central electrodes relative to the frontal electrodes. There was also a significant main effect of laterality  tat was the result of larger P2 amplitudes for the middle electrodes relative to the other electrodes (p < 0.02) and larger P2 amplitudes for the medial electrodes relative to the lateral electrodes (p < 0.03).A three-way RM-ANOVA conducted on the P2 amplitudes revealed a significant main effect of attentional load  and anteriority . However, P2 latencies were modulated as a function of laterality , as reflected by significantly longer P2 latencies at the lateral right electrodes relative to than the medial left (p = 0.002), lateral left (p = 0.012), medial right (p = 0.003), and middle electrodes (p = 0.001).For the P2 latencies, there were no significant main effects of attentional load [By asking participants to attend to pitch perturbations in their voice auditory feedback while concurrently performing a low-load, intermediate-load, and high-load visual attention task, the present cross-modal study investigated the auditory-motor processing of vocal pitch errors during divided attention. The behavioral results revealed significantly smaller vocal compensations for attended pitch perturbations in the low-load condition relative to the intermediate-load and high-load conditions. Differential effects of divided attention were observed on the cortical N1 and P2 responses to attended pitch perturbations. The high-load condition elicited significantly larger N1 responses and smaller P2 responses than the intermediate-load and low-load conditions. These findings provide behavioral and neural evidence that divided attention can modulate the auditory-motor processing of vocal pitch errors.In a previous study by Liu et al. , we showGiven that attentional capacity is limited  in working memory, should be conducted to verify our speculation.In summary, the present cross-modal study investigated the behavioral and neural correlates of auditory-motor integration for vocal pitch regulation during divided attention. The results revealed enhanced vocal compensations for pitch perturbations, enhanced N1 responses, and suppressed P2 responses with increasing load of divided attention, providing neurobehavioral evidence that divided attention can exert top-down influences on auditory feedback control of speech production. Considering the involvement of working memory in divided attention for the storage and maintenance of multiple sensory information (Johnson and Zatorre, HL and BZ: Designed the experiment; YL, HF, JL, and PL: Performed the experiment and analyzed the data; YL, JJ, BZ, and HL: Interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malathion (MLT) is an organophosphorous type pesticide and having seriously high toxicity and electrochemical platforms for rapid, simple, inexpensive and sensitive determination of pesticides is still a special concern. This paper describes a simple preparation of a composite film consisting of ionic liquid (IL), chitosan (CS) and electrochemically synthesized gold nanoparticles (AuNPs) on single use pencil graphite electrodes (PGEs). The microscopic and electrochemical characterization of AuNP-CS-IL/PGE was studied using scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. This fabricated surface was then explored for the first time as a sensing matrix for the non-enzymatic electrochemical sensing of malathion by cyclic voltammetry and square wave voltammetry measurements. The proposed AuNP-CS-IL/PGE showed excellent characteristics and possessed remarkable affinity for malathion. The voltammetric current response exhibited two linear dynamic ranges, 0.89–5.94 nM and 5.94–44.6 nM reflecting two binding sites, with a detection limit of 0.68 nM. The method was applied in real sample analysis of apple and tomato. The results demonstrate the feasibility of AuNP-CS-IL-modified electrodes for simple, fast, ultrasensitive and inexpensive detection of MLT. Organophosphorus pesticides (OPs) are an integral part of modern agriculture due to their high insecticidal activity against pests that are especially corruptive to crops and foods . OPs areS-bis (etoxycarbonyl) ethyl O,O-dimetyl phosphorodithioate), is a broad spectrum, nonsystemic insecticide belonging to the OP class. It is extensively used in public health programs against mosquito-borne illness, fruit fly eradication programs and indoor and outdoor residentals 3−/4−. Cyclic voltammetry is an efficient tool to monitor the kinetic barrier of the interface and the electron transfer between the solution species and the electrode [6]3−/4− couple was observed. The average anodic peak current (Ia) for the bare PGE was 55.12 ± 2.64 µA (n = 3). After the immobilization of CS-IL (curve b), peak currents increased (Ia = 78.52 ± 3.72 µA) and peak-to-peak potential difference decreased, indicating a faster electron-transfer could take place at CS-IL interface due to the conductive property of IL. When AuNPs were electrodeposited on this surface (curve c), the magnitude of the peak currents further increased (Ia = 137.2 ± 4.82 µA) and peak-to-peak separation (ΔEp) obviously decreased. This may be attributed to excellent catalytic activity of AuNPs. Moreover, the synergistic effect of composite and the large surface area contributed to the improvement in the conductivity of the AuNP-CS-IL modified electrode. The increase in surface area of the PGE after modifications were calculated using the Randles–Sevcik equation:−1), D is the diffusion coefficient of the analyte (cm2 s−1), 7.6 × 10−6 cm2 s−1 (at T = 298 K), and A is the working electrode area (cm2). The calculated electroactive surface area of the bare electrode is 0.066 ± 0.003 cm2 and AuNP-CS-IL coating on the surface significantly increased the surface area to 0.165 ± 0.006 cm2.The assembly process of the modified electrode and the electrochemical properties of different modified layers were monitored in 0.1 M KCl containing 5.0 mM [Fe(CN)lectrode . Figure Electrochemical impedance spectroscopy (EIS) was used in order to demonstrate the interfacial properties of the electrodes. Comparison of cyclic voltammogram and EIS spectrum of AuNP coated PGE in the absence of CS-IL were shown in As mentioned, for the electrochemical detection of MLT, the AChE inhibition approach has been most commonly applied by measuring the decrease of the oxidation current of thiocholine, the electroactive product of enzymatic reaction of AChE ,11,12,27Reduction behavior dithiophosphonates and -P = S- containing structures was also reported in the study of Cekirdek et al. , in whic−1 indicating that the reduction process on this modified electrode was a typical surface (adsorption) controlled process  technique, which yields higher sensitivity in electrochemical measurements. After accumulation of MLT on the sensor surface by stirring the solution for 300 s under open-circuit, the square wave voltammogram for the electroreduction of 3.0 nM MLT in BR buffer (pH = 7.0) at the AuNP-CS-IL/PGE was examined in 2 = 0.987) in the range of 0.89–5.94 nM and −Ipc (μA) = 0.5287 C (nM) + 19.657 (R2 = 0.968) in the range of 5.94–44.6 nM, respectively with a detection limit (LOD) of 0.68 nM. The LOD was calculated from 3 Sb/m, where Sb is the standard deviation of blank and m is the slope of the regression line.The comparison of the performance of the AuNP-CS-IL modified electrode with other reports based on enzyme inhibition and nanostructured materials is shown in The influence of possible interfering substances on the determination of MLT was studied where the concentration of MLT was fixed at 15 nM. The electrode response was examined for 1000-fold higher quantity of an organophosphorus pesticide, fenitrothion and 2 mM of some potential interfering ions in water. The results were displayed in To evaluate the suitability of the biosensor for real samples, recovery studies were carried out. Tomato and apple samples were spiked with different standard concentrations of MLT. The recoveries in the range of 91.1–114.2% were summarized in PGEs were easily modified with an AuNP-CS-IL hybrid to detect an organophosphorus pesticide, malathion directly, for the first time. The benefits of ILs (high conductivity and good adsorption for OPs), CS  and AuNPs  were combined on disposable pencil graphite electrodes in a synergistic effect. The nanocomposite film not only enhanced the surface area of the electrodes but also provided an excellent platform for obtaining the electrochemical response of malathion without the assistance of AChE. Square wave voltammetry has been applied in order to investigate the electrochemical performance of the AuNP-CS-IL/PGE based sensor towards malathion. The presented sensor was a promising sensing platform for the highly sensitive and fast determination of malathion, which is important for public health, environment environmental security and food safety. The proposed non-enzymatic approach was simple, efficient and cost-effective for the analysis of MLT in food samples and this method can be used as an alternative in the future.


Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, β-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, β-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. β-Glucosidase, β-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors  Glycosidases are key enzymes in central and intermediate metabolic pathways. They release terminal sugars from glycosides as oligo- or disaccharides, aryl, and alkyl glycosides. They may also have activity against a wide array of glycoconjugates, as polysaccharides, glycoproteins, or glycolipids . Alternatively, colorimetric or fluorogenic groups from synthetic substrates as Insect digestive glycosidases are essential enzymes for nutrition, regardless of taxonomic order or diet. These enzymes are essential for final digestion of sugars and have important roles in the interaction of insects with plants and pathogens  and Lutzomyia longipalpis (Diptera: Phlebotominae). We tested the use of continuous assays for the detection and measurement of acidic digestive glycosidases in these two insect species. Standardization of assay conditions allowed us to obtain continuous activity measurements similar to the discontinuous protocol. We applied those techniques for the measurement of six glycosidases  in R. prolixus and α-Glucosidase in L. longipalpis. Additionally, we conducted high-throughput measurements of these enzymes in R. prolixus, which allowed us to study simultaneously the population distribution of all these activities using individual insect samples.The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Saccharomyces cerevisiae (cat. No. G5003). All reagents used in this work are analytical grade (Sigma-Aldrich).The substrates used in this study were all from Sigma-Aldrich, namely 4-methylumbelliferyl α-D-glucopyranoside ; 4-methylumbelliferyl α-D-mannopyranoside ; 4-methylumbelliferyl β-D-glucopyranoside ; 4-methylumbelliferyl β-D-galactopyranoside ; 4-methylumbelliferyl N-acetyl-β-glucosaminide , and 4-methylumbelliferyl α-L-fucopyranoside . Stock solutions were prepared in dimethyl sulfoxide (DMSO) at 10 mM, α-glucosidase was from R. prolixus (Hemiptera: Reduviidae), obtained from our insectary at FIOCRUZ (Rio de Janeiro), maintained at 28°C and 75% relative humidity. Insects were regularly fed with defibrinated rabbit blood  were kept as described in Moraes et al. , Sodium Acetate (pH 4), MES (pH 5–7), Cacodylate (pH 8), Tris (pH 9), and Sodium Carbonate (pH 10–11), in the amount range from 62.4 to 624 picomoles.R. prolixus, we separated the Anterior Midgut (AM) and homogenized the whole organ in 0.6 mL 0.9% NaCl. After that, contents and tissues were separated by centrifugation at 4°C, 5,000 X g, for 5 min  and homogenized them in 50 μL of citrate phosphate buffer 50 mM pH 6 containing Triton X-100 1% (v/v) for better solubilization.Insects were immobilized by placing them on the ice and dissected in cold 0.9% (w/v) NaCl. For Ex = 355 nm and λEm = 460 nm. Glycosidase activities were expressed in micro units (μU) per insect. One unit (U) correspond to the enzyme quantity that process 1 μmol of the substrate for a minute . Substrate hydrolysis was monitored at 30°C in a spectrofluorometer Spectramax Gemini XPS (Molecular Devices) on λe IUBMB, . In contt-test, and data for enzymes that did not present Gaussian distribution were submitted to non-parametric Mann–Whitney tests.Graphs and data analysis were performed with the software GraphPad Prism version 5.01 for Windows. Lilliefors-van Soest and Shapiro–Wilks normality tests were employed for verification of Gaussian distributions. Data for activities that showed a normal distribution in the population were submitted to parametric unpaired Student's R. prolixus and L. longipalpis are routinely done at pHs 4.5 and 6, respectively. For that, we performed standard curves for MU in pHs ranging from 2 to 11  group might be detected in acidic pHs, because glycosidase assays for 1 Figure . We obse1 Figure . NeverthTo confirm if the release of the MU group by a glycosidase might be followed continuously in acidic conditions, we performed test assays with a commercial α-glucosidase (Sigma) in pH 6 Figure . All assR. prolixus anterior midgut tissue homogenates . We observed in the assays for R. prolixus β-glucosidase, β-galactosidase, and α-fucosidase an initial lag phase in the first 5 min but, after that, linearity was restored .Similarly to what was observed with the commercial α-glucosidase, we tested if assays using intestinal homogenates of y Figure . In the y Figure . Howevery Figure , β-glucoy Figure , β-galacy Figure , β-N-acey Figure , and α-fy Figure . SimilarRhodnius prolixus for the six glycosidases anteriorly standardized using both protocols. The detected activity amounts of α-glucosidase, α-mannosidase, β-glucosidase, β-galactosidase, β-N-acetyl-hexosaminidase and α-fucosidase activities obtained with the continuous assay were statistically similar to the activities detected using the discontinuous protocol . For doing that, we assayed the same samples of anterior midgut tissue homogenates of l Figure . A simile Figure . In facte Figure .R. prolixus anterior midguts and submitted individual tissue homogenates to the continuous glycosidase assays in the conditions established above. To avoid variations in activity due to different ages or nutritional status, we used synchronized insects that were kept starving for 40 days. The starvation was also performed with the intention of simulating the poor nutritional status that is common in field-collected triatomines. That allowed us to measure the activities of the different six glycosidases in all individual samples in few hours. Results are summarized in Figure After validation of the continuous protocol with our biological samples, we decided to apply the continuous technique to the high-throughput screening of glycosidase activities in individual insect samples. For that, we dissected 90 We decided to analyze the distribution of activities for each enzyme, to test if these activities have a normal distribution across the population. Frequency histograms are presented in Figure Another type of analysis that the high throughput assay allows is to observe the correlation between enzyme activities using a high number of individuals for the comparison. In general, all glycosidase activities showed a significant correlation to the other glycosidases assessed, based on Spearman correlation coefficients  showed mono-modal frequency distributions, suggesting that they were recorded in a relatively homogenous population. We had the concern of analyzing insects that were synchronized and starved for a long period, to avoid interferences due to different ages or nutritional status, which can severely affect the fitness and physiological characteristics of R. prolixus  the distinction between parametric or non-parametric is not critical for most analysis  and that it might be very interesting to corroborate them in the future with more extensive samplings.A straightforward consequence of the distributions observed is that for future studies, parametric tests for comparisons between samples and further analysis should be used only for the study of An interesting feature that the individual measurement of glycosidase activities allowed us to assess is the possibility of a correlation between enzymes when looking at different insects across the population. This approach allowed the observation of a positive correlation between all the measured glycosidase activities. We expect a basal level of correlation in all comparisons because all activities would be dependent on the protein content of samples. Nevertheless, unusually high levels of correlation between some glycosidases were registered, which might reflect physiological functional groups of enzymes working in concert against the same substrate. Of special interest is the grouping between β-glucosidase and α-fucosidase. We need further studies to understand the relevance of these correlations, both at the molecular and physiological levels.To our knowledge, this work is the first high-throughput screening of insect glycosidases performed at the individual level. It is important to notice that these enzymes are key factors in pathogen-vector and insect-plant interactions. Some glycosidases have been described as markers for insect resistance against plant defenses (Terra and Ferreira, Conception and design of the work: GP, CM, FG. Obtainment of experimental data: GP, JP, SC. Data analysis: GP, JP, SC, FG. Writing and revision of the manuscript: GP, JP, SC, PA, EG, CM, FG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MP declared a past co-authorship with several of the authors SC, PA, and EG to the handling Editor, who ensured that the process met the standards of a fair and objective review.


More than half of adults believed that O3-FAs are beneficial for heart and brain health and could correctly identify the food sources of O3-FA. However, the mean O3-I in the U.S. (4.3%) and Germany (5.5%) puts the majority of adults sampled (99%) in intermediate or high CVD-risk categories. More Americans were considered at high CVD-risk (40%) when compared with Germans (10%). In the U.S., but not Germany, women had a significantly higher O3-I than men . In the intermediate CVD-risk group, about one-third of adults in both countries (30% in the U.S. and 27% in Germany) believed their diet was adequate in O3-FA. Notably, mean O3-I concentrations did not significantly differ with dietary perceptions of adequacy. More adults in Germany (26%) than in the U.S. (10%) believed that dietary supplements are needed to achieve a balanced diet. In spite of adequate knowledge about food sources and a consistent belief that O3-FA are important for health, very few participants had O3-I concentrations in the range for CVD protection.Little is known about the relationship between perceptions of nutrient adequacy and biomarkers of nutrition status. This cross-sectional study of U.S. and German adults ( The Academy of Nutrition and Dietetics recommends adopting a balanced diet with a variety of nutrients for achieving optimal dietary intakes . UnfortuSeafood (both fish and shellfish) and dietary supplements are important sources of O3-FA . The 201Previously, a study on perceptions of healthy diet among adults showed that more individuals in the U.S. than Germany reported the use of O3-FA supplements (45% vs. 24%) . Most  were modified after a previous study . SpecifiWhole blood was collected using an ethylenediamine tetraacetic acid (EDTA) tube for testing for O3-I. Samples were centrifuged at 3000 rpm at room temperature for 15 minutes. Plasma and buffy coat were removed and the RBC pellets were stored at −80 °C. In the U.S., all samples were shipped to OmegaQuant Analytics , and in Germany all samples were sent to Omegametrix  on dry ice for testing for O3-I. Omega-3 index is a biomarker for O3-FA, which is calculated as a sum of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in the RBC membranes and reflects the long-term intake of EPA and DHA ,15,16. Ap-value < 0.05).Statistical analysis was performed using SAS statistical software 9.4 . Means and proportions were computed to describe the baseline characteristics of the individuals enrolled in the study. To compare the differences between sex and O3-I risk categories, an ANOVA  was used for continuous variables with normal distributions, non-parametric tests for continuous variables with non-normal distributions, and chi-square test for categorical variables (2) in both countries. Men had a higher BMI than women in both countries but no other sex differences were noted within each country. The majority of adults in both countries reported alcohol use and more participants in Germany were current smokers than in the U.S. Most adults rated improving or maintaining health as either important or very important  were, on average, older than participants in the U.S.  . Most pamportant .Almost all the participants (99%) reported that consuming a balanced diet is important, but only 50% from the U.S. and 41% from Germany thought that the food that they eat is nutritionally balanced (data not shown). More than half of the participants in the U.S. (69%) and in Germany (56%) believed that a balanced diet can be achieved through food alone and that dietary supplements are not required (data not shown). More adults in Germany (26%) than in the U.S. (10%) believe that dietary supplements are needed to achieve a balanced diet (data not shown).All participants believed that O3-FAs are important for health. Both in the U.S. and Germany, O3-FAs were considered important for improving heart health , overall wellness , and brain health . Almost all participants from the U.S. and Germany thought that oily fish  contain O3-FAs  . Howevern = 4). Only 10% of participants in Germany were classified as high risk for CVD compared with 40% of U.S. participants (data not shown). Women had a significantly higher O3-I than men in the U.S., while no such sex difference was observed in Germany.The mean O3-I values in the U.S. and Germany were 4.3% and 5.5%, respectively . None ofO3-I was not significantly different between people who perceived their diet as adequate in O3-FA and those who did not perceive their diet as adequate in both study sites . While cAlmost all individuals in our study, regardless of country, had a fair knowledge of the health benefits of O3-FAs and correctly identified the food sources of O3-FAs. All participants believed that a balanced diet is important for health; many acknowledged that a balanced diet can be achieved through food alone, with no need for dietary supplements. However, the O3-I biomarker status was below the optimal range in almost all individuals from both countries. This discrepancy may be due to a misconstrued interpretation of perceptions of what constitutes a nutritionally balanced diet or other factors that may prevent them from achieving the intakes for optimal status.In spite of having similar knowledge and perceptions about O3-FA, the mean O3-I was higher and the number of individuals classified as high risk for CVD was lower in Germany compared to the U.S. One possible reason for this discrepancy could be a greater consumption of fish products in Germany compared to the U.S. Secondly, even when consumed, not all fish contain sufficient amounts of EPA and DHA. Studies in the U.S. have shown that the concentration of EPA and DHA has decreased in farm grown fish across time . ConsumpSeafood is a major source of EPA and DHA, and it has been well established that maintaining higher blood concentrations of O3-FA is associated with reduced risk of CVD ,25,26,27The O3-I has been demonstrated to have a good dose-response relationship with dietary intakes of O3-FAs, and the O3-I has been a validated surrogate biomarker of dietary intakes ,36,37,38Some limitations of the current study exist, and our findings should be interpreted with these caveats in mind. First, our participants were recruited through a convenience sampling procedure and were relatively young and well educated. Due to small sample size, four individuals from Germany with O3-FA values in the ‘optimal range’ were included in the ‘intermediate risk for CVD category’. In the questionnaire, we defined a balanced diet as a ‘diet with all the recommended daily intake of nutrients’, however there is no general scientific definition for a balanced diet and it can be interpreted differently. Nevertheless, almost all participants in both countries felt consuming a balanced diet was important (99%).In spite of adequate knowledge of nutrients and their health effects, individuals did not have adequate O3-I levels. In order to provide appropriate recommendations on diet and dietary needs, future studies are required to identify factors that influence O3-FA intake, including cost, availability, and dietary preferences. Healthcare professionals and those responsible for helping individuals achieve higher levels of O3-FA need to take into account these realistic challenges and offer solutions for closing the gap between dietary intake and cardio protective levels of O3-FA, including but not limited to supplementation, if appropriate.